Enzymatic Nitrogen Insertion into Unactivated C-H Bonds

Abstract

Selective functionalization of aliphatic C-H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C-H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C-H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp³)-H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Figure 1.. Paths for enzymatic nitrogen functionalization of C–H bonds.

Path a, prevalent in extant biochemistry, utilizes oxygenation of C–H bonds as a first step. Cytochromes P450, for example, form a high valent Fe-oxo species and direct it for site- and stereo-specific oxygenation of unactivated C–H bonds. The oxygenated products can be further functionalized to nitrogen-containing compounds. In nature, such heteroatom C–H functionalization is restricted to oxygenation or halogenation. Path b depicts new-to-nature heme enzymes that catalyze C–H nitrene insertion reactions via formation of an active site Fe-nitrenoid, analogous to natural P450 chemistry’s Fe-oxo species. Previously reported ‘nitrene transferases’ showed activity only at electronically activated benzylic, allylic and propargylic positions. This work presents enzymes that use simple hydroxylamine derivatives 1 or 2 as nitrenoid precursors for selective nitrogen functionalization of unactivated C–H bonds to give primary amines or acetamides.

Figure 2.. Evolution of enzymatic C–H amination (A) and amidation (B).

Starting from parent enzymes uPA0 and uAMD0, directed evolution improved amination or amidation of 3 as assayed by product (5 or 7) yield. Reactions were set up with whole cells expressing the variant of interest at an optical density (OD) of 30 and 2.5% v/v EtOH. Amination reactions were performed with 2.5 mM of 3, 5 mM of 1, and amidation reactions with 2.5 mM of 3, 10 mM of 2. Enzymatic C–H functionalization results in the regiodivergent formation of the 1,3-isomer (6) or the 1,2-isomer (8) as the major product., *with respect to the previous variant, **combined analytical yield of all product isomers, Δ stop codon, § additional frameshift or silent mutations. Refer to text and Supplementary Information for further details.

Figure 3.. Substrate scope of enzymatic amination and amidation.

A. Quantitative determination of product distribution for functionalization of substituted hydrocarbons. ^a with uAPA9, ^b with uAMD9, ^c with uAMD6, ^d with uAMD8*, ^e with uAMD7-T438S B. A survey of competent substrates showing proof of functionalization (amination with uAPA9, amidation with uAMD8*; yields and site selectivity not determined). In all cases, reactions were performed under whole-cell conditions with uPA or uAMD variants at an optical density (OD) of 30, with 5 mM of substrate, 10 mM of 1 or 2, and 2.5 % v/v EtOH. (see Supplementary Information for details).

Figure 4.. Computational modeling aided mechanistic insights into enzyme-catalyzed amidation of methylcyclohexane.

A. Comparison of the DFT-computed free energy profiles and experimentally obtained kinetic isotope effects (KIEs) for the amidation of unactivated (methyl cyclohexane) and activated (ethyl benzene) substrates. B. Steric map analysis of the H-atom transfer steps (³TS_HAT) leading to the formation of the 2-, 3-, and 4-regioisomeric products, as obtained from QMMM computations simulated in uAMD8. C. Depiction of the diastereoselective radical rebound step (TS_CN) for the amidation of 3, obtained from MD simulations in uAMD8.